Application of OsCPN10a gene in regulating rice seed vigor, grain type and drought tolerance

Abstract

The application of OsCPN10a gene in regulating rice seed vigor, grain type and drought tolerance belongs to the technical field of rice genetic engineering, and specifically relates to the research of OsCPN10a gene in regulating rice seed germination rate, germination potential, germination index, grain length, grain width, 1000-grain weight and rice seed drought tolerance. The experimental results show that the grain type of OsCPN10a knockout mutant seed is obviously longer and wider, while the seed of OsCPN10a overexpression is opposite; the yield of Oscpn10a mutant is higher than that of TNG67 plant; OsCPN10a overexpression has the potential to improve the drought resistance of rice. The application discloses the genetic mechanism of OsCPN10a affecting rice seed vigor, grain length, grain width and storage resistance, provides important gene resources and theoretical basis for genetic improvement of rice, and helps to cultivate new rice varieties with better grain type and higher yield.

Description

Technical Field

[0001] This invention belongs to the field of rice genetic engineering technology, and particularly relates to the application of the OsCPN10a gene in regulating rice seed vigor, grain shape and drought resistance. Background Technology

[0002] Seed germination begins with water absorption and swelling, followed by the recovery of seed metabolism. The radicle breaks through the endosperm and seed coat to complete germination. The quality of seed germination hinges on the quality of stored mRNA, protein stability, and DNA integrity. During germination, intracellular stored substances such as fats, proteins, and soluble sugars are broken down to synthesize new macromolecules, primarily proteins and nucleic acids, along with energy consumption. The level of stored protein in the seed directly affects seed germination and seedling growth, providing a source of nitrogen for normal metabolism and development.

[0003] Molecular chaperones, also known as heat shock proteins (HSPs), are found in the plastids, mitochondria, and cytoplasm of all eukaryotes and bacteria. They are a class of proteins that help other proteins fold and assemble correctly, refold denatured proteins, prevent misfolding, and, when necessary, unfold or degrade abnormal proteins to maintain cellular protein homeostasis, which is essential for cell survival. Molecular chaperones play a crucial role in maintaining protein homeostasis, participating in various biological processes, including protein synthesis, transmembrane transport, assembly and disassembly of protein complexes, and responses to cellular stress. In plants, molecular chaperones participate in various metabolic pathways, playing important roles in plant growth and development, metabolic regulation, and abiotic stress. They are mainly divided into two different types: type I molecular chaperones and type II molecular chaperones. Type I chaperone proteins (such as HSP110, HSP100, HSP90, HSP80, Hsp70, Hsp60, and Hsp40, and CPN60) often function in conjunction with co-chaperone proteins. Plant plastid and mitochondrial co-chaperone proteins include Cpn21 and Cpn10. The co-chaperone protein for HSP60 is HSP10; the co-chaperone proteins for Cpn60 are Cpn10 (10 kDa chaperonin) and CPN20. Cpn60 proteins are crucial for plants because they participate in the folding of many chloroplast proteins and polypeptides. Kaur et al. identified the OsHSPl8.2 gene using omics, whose main function is to influence rice seed vigor, lifespan, and seedling formation by regulating the accumulation of reactive oxygen species (ROS). Currently, little is known about the functions of co-chaperone protein family members in rice.

[0004] 10-kDa co-chaperone protein (CPN10) is a common co-chaperone protein in plant plastids and mitochondria, with a molecular weight of approximately 10 kDa, similar to Gro ES in bacteria, and can form a heptamer ring structure. CPN10 is a multifunctional protein, and Arabidopsis includes CPN10(1), CPN10(2), Plastid CPN10(1), and Plastid CPN10(2), which play an important role in regulating mitochondrial and chloroplast structure maintenance. Zheng et al. knocked down Cpn10(1) in Arabidopsis mitochondria, leading to mitochondrial dysfunction, further confirming the importance of CPN10 in maintaining mitochondrial homeostasis. Hemmingsen et al. found that Arabidopsis mitochondrial CPN10(1) and rice mitochondrial CPN10 had a similarity of 67%. It is known that the monocotyledonous model plant rice has two OsCPN10 genes, but whether these genes are involved in the rice's response to biotic and abiotic stresses has not been reported. Therefore, it is of great significance to explore the function and regulatory mechanism of rice OsCPN10 genes. Summary of the Invention

[0005] This invention uses wild-type, knockout, and overexpression rice lines as materials and combines plant physiology, biochemistry, molecular biology, and other techniques to preliminarily explore the function of OsCPN10a protein in biological processes such as rice response to seed germination, artificial aging, and drought stress, providing assistance for further elucidating the molecular mechanism of OsCPN10 in rice response to aging stress.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the application of the OsCPN10a gene in regulating rice seed vigor, grain shape and drought resistance. The gene knockout or gene overexpression regulates rice seed vigor, grain shape and drought resistance. The amino acid sequence of the protein encoded by the OsCPN10a gene is shown in SEQ ID NO.1, and the nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.2.

[0008] The regulation of seed activity refers to the regulation of seed germination rate, germination potential, and germination index; the regulation of seed grain shape refers to the regulation of seed grain length, grain width, and thousand-grain weight.

[0009] The gene knockout was performed using OsCPN10a as the target gene. A knockout vector for the target gene was constructed using the CRISPR / Cas9 gene editing system, transformed into rice, and then the rice was cultivated.

[0010] The method for constructing the knockout vector is as follows:

[0011] A target site was selected and gRNA was designed. PCR amplification was performed using pCBC-MT1T2 plasmid as a template. After recovery, the gRNA was ligated with pHUE411 vector using T4 ligase to obtain the ligation product. The product was then transformed into E. coli, the plasmid was extracted, and sequencing was performed for verification.

[0012] The gRNA sequence is shown in SEQ ID NO.3 or 4.

[0013] The gene overexpression method uses OsCPN10a as the target gene, and employs seamless cloning and homologous recombination to connect the target gene with the vector to construct an overexpression vector, which is then transformed into rice for rice cultivation.

[0014] The method for constructing the gene overexpression vector is as follows:

[0015] Upstream and downstream primers were designed for PCR amplification of the OsCPN10a gene. The binary vector pRHVcGFP and the amplification product were digested with HindIII and KpnI as restriction sites. Then, the target fragment was ligated to the linearized vector through homologous recombination to obtain the recombinant vector.

[0016] The upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.12.

[0017] Secondly, the present invention provides the application of the OsCPN10a gene in the breeding of drought-resistant rice varieties, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0018] Thirdly, the present invention provides a method for breeding drought-resistant rice varieties, wherein an expression vector containing the OsCPN10a gene is transformed into a target plant for overexpression to obtain the drought-resistant rice; the nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.2.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] To elucidate the function of OsCPN10 in rice, OsCPN10 (Os03g0366000) was cloned from rice embryos to construct knockout and overexpression transgenic lines. Using wild-type, knockout, and overexpression rice lines as materials, this paper employs plant physiological and biochemical methods, as well as molecular biology techniques, to preliminarily explore the function of OsCPN10a protein in biological processes such as rice response to seed germination, artificial aging, and drought stress. The results are as follows:

[0021] (1) There are certain differences in morphological characteristics between wild-type and transgenic rice lines. TNG67, Oscpn10a, and OE show significant differences in grain length and width. The OsCPN10a knockout mutant has significantly longer and wider grains, while the OsCPN10a overexpression seeds show the opposite. In the field, the Oscpn10a mutant yielded higher yields than TNG67 plants.

[0022] (2) This invention found that knocking out OsCPN10a leads to delayed seed germination and slow seedling growth, while overexpression of OsCPN10a results in earlier seed germination compared to the wild type. After artificial aging treatment, the germination rate of OsCPN10a seeds was significantly reduced. This indicates that the function of OsCPN10a is essential for normal early seed germination. During seed germination, the amount of ABA accumulated in OsCPN10a knockout mutant seeds at 8 hours of germination was 41% higher than that in TNG67. Under ABA treatment, the germination rate, root length, and shoot length of OsCPN10a seeds were significantly reduced, indicating that OsCPN10a has a negative regulatory effect on ABA accumulation. Compared to wild-type plants, the increased ABA accumulation in the OsCPN10a knockout mutant was associated with increased expression of ABA biosynthesis genes ZEP1, NCED1, NCED3, NCED4, and NCED5. Further analysis revealed that the expression levels of ABA marker genes NCEDs and ABIs in the knockout lines were higher than those in the wild type, while the expression levels of NCEDs and ABIs in the overexpression lines were lower than those in the wild type, indicating that OsCPN10a plays a negative regulatory role in the ABA signal transduction pathway.

[0023] (3) Under drought stress, the survival rate of OsCPN10a overexpressing seedlings was significantly higher than that of wild-type plants, while the survival rate of Oscpn10a knockout mutants was significantly lower than that of wild-type plants. The results indicate that OsCPN10a overexpression has the potential to improve the drought resistance of rice.

[0024] In summary, through molecular biology and genetics research, the genetic control mechanism by which OsCPN10a affects rice seed vigor, grain length, grain width, and storage tolerance has been revealed, providing important gene resources and a theoretical basis for the genetic improvement of rice. These research findings will contribute to the breeding of new rice varieties with better grain shape and higher yield. Attached Figure Description

[0025] Figure 1The results of OsCPN10a knockout and overexpression in the TNG67 background material in Example 4 are shown in Figure A, which is a schematic diagram of the OsCPN10a genome; Figure B is the OsCPN10a protein sequence alignment of TNG67 and OsCPN10a knockout lines; Figure C is a plant photograph of TNG67, Oscpn10a mutant and OsCPN10a overexpression line; Figure D is the relative expression analysis of OsCPN10a gene in TNG67 and OsCPN10a overexpression line.

[0026] Figure 2 The effect of the OsCPN10 mutant on seed grain shape in Example 5 (wherein, Figure A shows mature seeds of TNG67, Oscpn10a mutant and OsCPN10a overexpression line, Bar = 5 mm; Figure BG shows grain width, grain length, thousand-grain weight, single-plant grain yield, seed setting rate and single-plant effective tiller number of TNG67, Oscpn10a mutant and Oscpn10a overexpression line; Figure H shows scanning electron microscopy observation of the outer surface of mature seed glume, Bar = 100 μm; Figure IK shows cell length, width and number analysis; Figure LN shows the relative mRNA expression levels of OsDEP1, OsGS3 and OsGW2 in the embryos of mature seeds of TNG67, Oscpn10a mutant and OsCPN10a overexpression line).

[0027] Figure 3 Functional analysis of the OsCPN10a gene during seed germination in Example 6 (where Figure A shows seed germination photographs of TNG67, Oscpn10a knockout mutants and overexpression lines; Figures BD show a comparison of seed germination potential, germination rate and germination index of TNG67, Oscpn10a knockout mutants and overexpression lines).

[0028] Figure 4 This is a comparison of the seed vigor of TNG67, Oscpn10a mutant and Oscpn10a overexpression line rice under artificial aging in Example 6 (wherein, Figure A shows the seed germination of TNG67, Oscpn10a mutant and Oscpn10a overexpression line after 7, 14 and 21 days of artificial aging treatment at 14 days; Figure B shows the comparison of the germination potential, germination rate and germination index of TNG67, Oscpn10a mutant and Oscpn10a overexpression line seeds after 7, 14 and 21 days of artificial aging treatment; Figure C shows the appearance of TNG67, Oscpn10a mutant and OE line rice after 21 days of artificial aging, Bar = 100 μm; Figure D shows the cross-sectional electron micrographs of TNG67, Oscpn10a mutant and OE line rice after 21 days of artificial aging, Bar = 10 μm & 5 μm).

[0029] Figure 5This is a comparison of the seed vigor of TNG67, Oscpn10a mutants and Oscpn10a overexpression lines under natural aging conditions in Example 6 (wherein, Figure A shows the germination status of TNG67, Oscpn10a mutants and Oscpn10a overexpression lines 10 days after natural aging for 6 months; Figure B shows a comparison of the germination potential, germination rate and germination index of TNG67, Oscpn10a mutants and OE line seeds 10 days after natural aging for 6 months).

[0030] Figure 6 This study investigates the drought resistance of OsCPN10a and rice in Example 7 (Figure A shows the growth of the Oscpn10a knockout mutant, OE line, and TNG67 under normal nutrient solution culture and the phenotypic characteristics after 3 weeks of drought treatment followed by 3 days of rehydration, Bars = 10 cm; Figure B shows the statistical analysis of plant survival rates of TNG67, Oscpn10a knockout mutant, and OE line after 3 weeks of drought treatment followed by 3 days of rehydration; Figure CG shows the antioxidant activity of rice leaves of TNG67, Oscpn10a knockout mutant, and OE line before and after drought stress). Detailed Implementation

[0031] To better illustrate the present invention, the following embodiments are provided. Obviously, the described embodiments are merely a part of the present invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are all within the scope of protection of the present invention.

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0033] The amino acid sequence of the protein encoded by the rice OsCPN10a gene is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0034] SEQ ID NO.1:

[0035] MAARRLIPSMNRVLVEKLLQPNKSAGGILLPETTKQLNSAKVVAVGPGERDR DGKLIPVSLKEGDTVLLPEYGGTEVKLAEKEYLLFREHDILGRLEE*

[0036] SEQ ID NO.2:

[0037] ATGGCGGCGAGGAGGCTGATCCCGTCGATGAACCGGGTGCTGGTGGAGAA

[0038] GCTGCTGCAGCCCAACAAGAGCGCCGGCGGCATCCTCCTCCCGGAGACCA

[0039] CCAAGCAGCTGAATTCTGCAAAAGTAGTGGCTGTTGGTCCTGGCGAACGT

[0040] GACAGGGATGGCAAACTGATCCCTGTATCTTTGAAAGAAGGTGACACCGTT

[0041] CTGCTACCTGAGTATGGAGGAACTGAAGTGAAGCTTGCTGAGAAAGAGTA

[0042] CCTTCTTTTCAGAGAGCACGACATACTCGGACGGCTTGAGGAGTAA

[0043] Example 1: Knockout of the OsCPN10a gene based on the CRISPR / Cas9 technology

[0044] To create transgenic plants with clustered regular-spaced short palindromic repeats (CRISPR)-OsCPN10a knockout of the OsCPN10a gene, a single guide RNA (sgRNA) sequence for OsCPN10a, i.e., a CRISPR / Cas9 specific target site, was designed using the CRISPR-PLANT online website. Based on the results of specific target prediction, two gRNA targets were selected in the first exon of the OsCPN10a gene: target-1: gtcgatgaaccgggtgctgg (SEQ ID NO.3) and target-2: cctgcttggtggtctccggg (SEQ ID NO.4). Off-target analysis of the selected targets was then performed using the CRISPRRGEN Tools website. The designed knockout primers are as follows: Cpn10-F: AATATGGTCTCAGGCGTCGATGAACCGGG TGCTGGGTTTTAGAGCTAGAAATAGC (SEQ ID NO.5); PCBC-MT1-R (general): ATTATGGTCTCTGCTTCTTGGTGCCGC (SEQ ID NO.6); CPN10-MT2-F: ATAATATGGTCTCaAAGCCCCGGAGACCACCAAGCAGGGTTTTAGAGCTAGA AATAGC (SEQ ID NO.7); PCBC-MT2(m6b)-R (general): AATATGGTCTC aAACACAAGCGGCAGCGCGCG (SEQ ID NO.8).

[0045] Amplification of the gRNA target ligation fragment was performed using a 100-fold diluted pCBC-MT1T2 plasmid (laboratory stock, documented in the following literature: https: / / link.springer.com / article / 10.1186 / s12870-014-0327-y) as a template. Primers containing the BsaI restriction site were amplified using 2× Primer Star Max high-fidelity enzyme. After determining the fragment size, the fragments were recovered by gel extraction. The PCR reaction system and procedure are shown in Tables 1 and 2 below:

[0046] Table 1 PCR reaction system

[0047]

[0048] Table 2 PCR reaction procedures

[0049]

[0050] The amplified fragments were recovered and ligated into a cloning vector.

[0051] An enzyme digestion-ligation system was established using the pHUE411 vector to obtain the ligation product. The enzyme digestion and ligation reaction system and reaction procedure are shown in Tables 3 and 4 below:

[0052] Table 3 Enzyme digestion and ligation reaction system

[0053]

[0054]

[0055] Table 4. Enzyme digestion and ligation reaction procedures

[0056]

[0057] The ligation product was transformed into competent E. coli cells. The sequencing primers were universal primers for the pHUE411 vector. The specific primer sequence information is as follows: OsU3-FD3: GACAGGCGTCTTCTACTGGTGCTAC (SEQ ID NO.9); TaU3-RD: CTCACAAATTATCAGCACGCTAGTC (SEQ ID NO.10).

[0058] Extraction of recombinant plasmids: The correctly sequenced colonies were picked into 5 mL of LB liquid medium containing Kan antibiotic and cultured overnight. The recombinant plasmids were extracted using a plasmid extraction kit and then transformed into Agrobacterium competent cells EHA105.

[0059] Example 2: Overexpression of the OsCPN10a gene based on CRISPR / Cas9 technology

[0060] To construct the OsCPN10a overexpression vector, primers CPN10-Pro-F: ttgaaacactcccagtgatattagg (SEQ ID NO.11) and CPN10-Pro-R: cgacgggat cagcctcct (SEQ ID NO.12) were designed according to the method in Example 1. The full-length coding sequence of OsCPN10a was amplified from the Nipponbare genome, the target fragment was purified and recovered, and it was cloned into the binary vector pRHVcGFP (WGL28) (from the following literature: https: / / link.springer.com / article / 10.1186 / s12284-018-0220-7, the original name is pRHVcGFP) through seamless cloning, that is, the recombinant pUBI:OsCPN10a-GFP plasmid.

[0061] The pRHVcGFP(WGL28) vector was selected with HindIII and KpnI as restriction sites, and the vector plasmid was double-digested. The vector digestion reaction system is shown in Table 5 below:

[0062] Table 5 Enzyme digestion reaction system

[0063]

[0064]

[0065] The enzyme digestion conditions were 37°C for 3.5 hours or overnight. The digestion products were subjected to 1% agarose gel electrophoresis and then recovered by gel excision.

[0066] The target fragment was ligated to a linearized vector using homologous recombination, and the reaction system is shown in Table 6 below:

[0067] Table 6 Connection Reaction System

[0068]

[0069] The ligation conditions were 37°C for 30 minutes. Transformation was performed according to the transformation system and steps described in Example 1, using cpn10-ver-F: CCCGTCACCCACTCACCCGTCT (SEQ ID NO.13) and cpn10-ver-R: CACCGCTCTATGTCCCAAACCTC (SEQ ID NO.14). After successful sequencing, the recombinant plasmid was extracted according to the kit instructions and transformed into Agrobacterium competent cells EHA105.

[0070] Example 3 Agrobacterium-mediated genetic transformation of rice callus

[0071] All transgenic rice plants were obtained through Agrobacterium-mediated transformation of rice callus.

[0072] (1) Induction and subculture of callus

[0073] Preparation of materials: Select about 100 freshly harvested, plump seeds (TNG67), carefully remove the shells and set aside. Sterilize the Erlenmeyer flask, double-distilled water, and filter paper before use.

[0074] Disinfection: Transfer the prepared seeds to a sterilized Erlenmeyer flask, rinse the seeds 4-5 times with sterile distilled water until no impurities are present, then disinfect the seed surface with sterile 75% ethanol for 2 minutes, followed by sterilization with 2.5% sodium hypochlorite in a shaker at room temperature for 20 minutes. Then rinse with sterile water until no sodium hypochlorite residue remains, transfer to a petri dish containing filter paper and air dry.

[0075] Callus induction and subculture: The dried seeds were transferred to NEB induction medium and placed in a 28℃ light incubator to induce callus for about 12 days. After that, the buds were removed and the callus was subcultured. Subculture was carried out every 14 days thereafter (the time can be adjusted according to the state of the callus). In the late second generation, the bright yellow callus can be selected for Agrobacterium infection.

[0076] (2) Agrobacterium-mediated transformation of rice callus

[0077] Strain activation: Take an appropriate amount of the strain stored at -80℃ and streak it onto a solid medium containing Rif and Kan antibiotics, and incubate in the dark at 28℃ for 2 days.

[0078] Pre-culture of callus: Select bright yellow rice callus in good condition and culture it in NEB medium at 28℃ for 4 days.

[0079] Agrobacterium infection: The bacterial cells were washed with sterile AAM, and the washed-off bacterial cells were transferred to sterile 50 mL centrifuge tubes. The OD of the bacterial solution was measured using a spectrophotometer, and the OD of the bacterial solution was adjusted using AAM. 600 The concentration was 0.3, and the culture was placed in the dark at 28°C for 1 hour. The pre-cultured callus (4 days) was then transferred to an Agrobacterium tumefaciens culture containing overexpression / knockout plasmids. The infection time was 5-10 minutes, with gentle shaking several times during the process. After infection, the infected callus was transferred to a culture dish containing several sheets of filter paper. Excess bacterial solution was blotted out, and the callus was then transferred to a culture dish lined with 1-2 sheets of filter paper. The callus surface was allowed to dry completely before being transferred to a co-culture medium and co-cultured in the dark at 28°C for 1 day.

[0080] Screening and differentiation of resistant callus into seedlings: Callus cultured in the dark for 1 day was transferred to a first-generation selection medium containing Carb and Hyg, with a generation selection every 14 days. New callus will grow during the selection process. Once the new callus has grown to a certain size, it is transferred to a pre-differentiation medium and cultured under light at 28°C until seedlings emerge. When the seedlings reach 3-4 cm in length and contain roots, they are transferred from the pre-differentiation medium to a rooting medium and cultured under light for a further period.

[0081] Hardening off seedlings: Once the seedlings have developed a strong root system, transplant them into a nutrient solution to harden off.

[0082] Example 4: Screening of transgenic seedlings

[0083] For the detection of transgenic seedlings, when the transgenic plants grew to 5 leaves, DNA was extracted from the leaves using the CTAB method. After PCR amplification with specific primers, the DNA was sent to the company for sequencing. Positive plants were transplanted into peat moss and cultivated in a greenhouse for 3.5 months to harvest T0 generation transgenic rice seeds. The obtained T0 generation transgenic rice seeds were then cultivated in field soil, and genomic DNA was extracted again from leaves at the same location from all lines.

[0084] The OsCPN10a knockout mutant was amplified by PCR using specific primers cpn10-ver-F: CCCGTCACCC ACTCACCCGTCT (SEQ ID NO.15) and cpn10-ver-R: CACCGC TCTATGT CCCAAACCTC (SEQ ID NO.16) to obtain DNA from seeds of generation T0 and leaves of subsequent generations, followed by sequencing analysis. The sequencing results were compared with the original sequences using SnapGene software to identify positive transformation materials and screen rice lines with mutations or deletions at the target gene sites for subsequent experiments.

[0085] The copy number of transgenic plants overexpressing the vector was analyzed by qRT-PCR using primers conum-sfu2af-F / conum-sfu2af-R and conum-HYG-F / conum-HYG-R. DNA was also detected in seeds of generation T0 and leaves of subsequent generations. Specific sequence information is shown in Table 7 below.

[0086] Table 7 Sequence Information

[0087]

[0088] Experimental Results: To investigate the function of the OsCPN10a gene, we constructed OsCPN10a knockout mutants and overexpression transgenic lines using CRISPR / Cas9 with Tainong 67 as the background. See details below. Figure 1Through propagation, we obtained four T2 generation homozygous OsCPN10a knockout lines: Oscpn10a-1, Oscpn10a-2, Oscpn10a-3, and Oscpn10a-4. All mutation sites were located in the first exon of the OsCPN10a gene. Oscpn10a-1 showed a one-base "G" insertion at the first target site of OsCPN10a; the Oscpn10a-2 mutant showed an "A" insertion at the first target site and an 11bp deletion (SEQ ID NO.21: -CTCCTCCCGGA-) at the second target site; Oscpn10a-3 showed "G" deletions at both the first and second target sites of OsCPN10a; and Oscpn10a-4 showed a 52bp deletion in the first exon of OsCPN10a. Based on these nucleotide sequence predictions, the amino acid sequence of OsCPN10a in Oscpn10a-1, Oscpn10a-2, Oscpn10a-3, and Oscpn10a-4 contains only 61, 28, 60, and 53 amino acids, respectively. These mutations all lead to premature termination of OsCPN10a translation. These results indicate that these four Oscpn10a mutants may lack a functional OsCPN10a protein.

[0089] Compared with TNG67, the mRNA expression level of OsCPN10a increased by 29-fold and 92-fold in the OsCPN10-OE1 (OE-1) and OsCPN10-OE2 (OE-2) overexpression lines, respectively.

[0090] The offspring of these homozygous mutants were used in subsequent experiments.

[0091] Example 5: Observation of mutant phenotypes

[0092] Seeds of wild-type TNG67, mutants Oscpn10a-2 and Oscpn10a-4, and transgenic rice with different overexpression lines were selected and planted in the field. Agronomic traits such as grain length, grain width, and yield of rice were evaluated at the tillering and maturity stages.

[0093] Experimental Results: Seed shape and size are key factors affecting yield and are also target traits for genetic engineering and molecular breeding. Studying the genetic effects of seed shape is of great significance for high-yield rice breeding. This study analyzed the influence of the OsCPN10a gene on yield, as detailed below. Figure 2The results showed that the seeds of the Oscpn10a knockout mutants were significantly longer. Compared with wild-type TNG67, the seed length of the Oscpn10a-2 and Oscpn10a-4 knockout mutants increased by 6.55% and 8.23%, respectively, and the seed width increased by 7.96% and 9.61%, respectively, while the seed length of OE-1 and OE-2 decreased by 12.41% and 6.55%, respectively, and the seed width decreased by 11.81% and 11.74%, respectively. The rice grains after removing the husk also exhibited a similar phenotype to the seeds. The thousand-grain weight of OE-1 and OE-2 decreased by 8.21% and 10.92% compared with TNG67, respectively, while the thousand-grain weight of Oscpn10a-2 and Oscpn10a-4 both increased by approximately 10%. Furthermore, yield data per plant showed that, under field conditions, overexpression or knockout of OsCPN10a led to either a decrease or increase in yield per plant. Among them, the yield per plant of Oscpn10a-2 and Oscpn10a-4 increased by 14.54% and 11.71%, respectively, while the yield per plant of OE-1 and OE-2 decreased significantly by 17.33% and 22.16%, respectively. To reveal the possible reasons for the changes in seed grain shape, the outer surface of the glumes of mature seeds was observed using scanning electron microscopy (SEM). The results showed that the cell length and width of the glumes in seeds overexpressing OsCPN10a were significantly reduced, while the cell length and width of the glumes in seeds of Oscpn10a-2 and Oscpn10a-4 were significantly increased. Compared with TNG67 plants, there were also significant differences in the total number of cells longitudinally on the outer surface of the glumes in Oscpn10a-2 and Oscpn10a-4 knockout mutants and the OE line. These results indicate that Oscpn10a negatively regulates seed grain length and width.

[0094] Example 6: Viability evaluation of OsCPN10a mutant seeds

[0095] Seeds from TNG67 and OsCPN10a knockout mutants and overexpression lines were germinated at room temperature under natural conditions, and after artificial aging treatment for 0, 7, 14, and 21 days, as well as after natural storage for six months. Seed storage tolerance and vigor were analyzed. Seed moisture content before and after aging was analyzed using a Risdec moisture analyzer, and apparent amylose (AAC) content and protein content were determined using reagent kits from Solarbio.

[0096] To observe the changes in seed structure before and after aging of OsCPN10a knockout and overexpression transgenic seeds, scanning electron microscopy was used to observe the cross-sections of the seeds. Specific procedures: The samples were fixed in 2.5% (w / v) glutaraldehyde (in 0.1M PBS) for 2 h, then washed three times with 0.1M PBS. After gradient dehydration with ethanol at 4°C, the samples were critically dried, coated with palladium using a sputtering coater, and then observed under a scanning electron microscope (JSM-6390LV, JEOL).

[0097] Experimental Results: Seed vigor is a key factor affecting seed germination and lifespan. This study evaluated the seed vigor of mature seeds from OsCPN10a knockout mutants and OsCPN10a overexpression lines. (See details...) Figure 3-5 The results showed that, compared with TNG67, the GP and GI of Oscpn10a-2 and Oscpn10a-4 seeds were significantly decreased after 3 days of germination, while the GP and GI of OE-1 and OE-2 seeds were significantly increased (P<0.05). Specifically, the GP of Oscpn10a-2 and Oscpn10a-4 seeds decreased by 35.48% and 62.37%, respectively, while the GP of OE-1 increased by 17.20%. However, there was no significant difference in the germination rate observed at 7 days among TNG67, Oscpn10a knockout mutant, and OsCPN10a overexpression line (P>0.05).

[0098] After 21 days of artificial aging, the germination rate, germination potential, and GI of Oscpn10a-2 and Oscpn10a-4 seeds were significantly lower than those of TNG67 seeds, while the germination rate, germination potential, and GI of OE-1 and OE-2 seeds were significantly higher than those of TNG67 (P<0.05). Specifically, the germination rate of Oscpn10a-2 and Oscpn10a-4 seeds aged for 21 days decreased by 65.13% and 67.61% respectively compared to TNG67, while the germination rates of OE-1 and OE-2 increased by 71.88% and 75.00% respectively.

[0099] A comparison of the appearance of polished rice from TNG67, Oscpn10a mutant, and OsCPN10a overexpressing transgenic seeds aged for 21 days revealed that OE-1 rice had relatively normal transparent grains, TNG67 had a semi-transparent dark endosperm phenotype, while Oscpn10a-4 was completely opaque milky white. Scanning electron microscopy analysis of the polished rice cross-sections showed that before artificial aging, the starch granules in the endosperm of TNG67, Oscpn10a-4, and OE-1 seeds were regular polyhedrals of uniform size and tightly packed. After 21 days of artificial aging, compared with TNG67 seeds, the starch granules in the endosperm of Oscpn10a-4 seeds were less uniform in shape and size, and their arrangement was also looser.

[0100] After 6 months of storage under natural conditions, compared with the wild type, the germination potential, germination rate, and GI of Oscpn10a mutant seeds were significantly reduced (P<0.01), while the germination potential, germination rate, and GI of OE-1 and OE-2 were significantly increased (P<0.05). These results indicate that OsCPN10a knockout and overexpression transgenic seeds showed similar trends under both natural and artificial aging conditions. These results suggest that OsCPN10a plays an important role in regulating seed vigor and lifespan.

[0101] The appearance of rice is influenced by various factors, such as moisture content, apparent amylose (AAC) content, and protein content. This study measured the moisture content of polished rice samples from TNG67, Oscpn10a mutants, and the OE line after artificial aging. The results showed that after 21 days of artificial aging, the moisture content of all samples was approximately 12%, with no significant difference (P>0.05). Analysis of the AAC content of different samples revealed that after 21 days of artificial aging, the AAC content of OE-1 and OE-2 rice was lower than that of the wild type, while the AAC content of Oscpn10a-2 and Oscpn10a-4 mutant rice was increased by 20.64% and 36.12% respectively compared to the wild type (Table 8), which is consistent with the dark endosperm phenotype of rice. Further analysis of the total protein content of different samples after 21 days of artificial aging revealed no significant difference in total protein content among TNG67, Oscpn10a mutant, and OE line rice (P>0.05). These results suggest that the differences in transparency among TNG67, Oscpn10a mutant, and OE line rice may be directly related to AAC, but not directly related to moisture content and protein content.

[0102] Table 8. AAC and total protein content of TNG67, Oscpn10a mutant, and OE line polished rice.

[0103]

[0104]

[0105] Data represent the mean ± SD (n = 3) from three independent replicates. In the table, the same lowercase letter indicates no significant difference compared to TNG67 under the same treatment, while different lowercase letters indicate significant differences compared to TNG67 under the same treatment. Student's t-test analysis was used to determine statistical significance. The same applies below.

[0106] Example 7: Drought stress treatment of OsCPN10a transgenic rice seedlings

[0107] In the soil drought stress treatment, wild-type and transgenic seeds that had germinated uniformly were transplanted into well-mixed soil (forest soil: vermiculite, ratio 1:1) and grown for 4 weeks under normal watering conditions. Then, a drought stress treatment was applied, with irrigation stopped for approximately 21 days. When all leaves curled, watering was resumed for 10 days. The survival rate of each plant was calculated (the ratio of surviving plants to the total number of treated plants in the pot). Simultaneously, stems and leaves from all plants on day 5 of the drought treatment were collected to assess antioxidant levels.

[0108] Diaminobenzidine (DAB) is the most sensitive and commonly used chromogenic substrate for horseradish peroxidase. In plant leaves, due to the presence of peroxidase, DAB reacts with H₂O₂ to form a yellowish-brown insoluble precipitate that accumulates on the leaf surface. The darker the color, the higher the H₂O₂ content. Nitroblue tetrazolium (NBT) staining is a commonly used histochemical method for detecting superoxide anion (O₂) in plant tissues. 2- In this experiment, DAB and NBT staining methods were used to detect H2O2 and O2 in the leaves of wild-type, OsCPN10a knockout mutant, and OsCPN10a overexpressing seedlings after 5 days of simulated drought treatment. 2- The content of.

[0109] Experimental results (see) Figure 6 Since OsCPN10a is ABA-induced and actively participates in the ABA response during post-germination growth, it is hypothesized that the response of Oscpn10a mutants and OsCPN10a overexpressing plants to drought stress may be altered. To test our hypothesis, we examined the drought stress response of soil-grown Oscpn10a mutants and OsCPN10a overexpressing plants. Wild-type, Oscpn10a-2, Oscpn10a-4, OE-1, and OE-2 plants were grown in soil under normal growing conditions for 4 weeks, followed by drought treatment. After 21 days without watering, Oscpn10a mutant plants exhibited a severe wilting phenotype, while wild-type and OsCPN10a plants grew well. After rehydration, only 25.0% of Oscpn10a-4 mutant plants survived, while the survival rate of OE-2 plants exceeded 78.0%. Significant differences were observed in stomatal development between wild-type and transgenic rice lines. Compared to the wild type, the leaves of Oscpn10a-2 and Oscpn10a-4 have closed stomata, while the leaves of OE-1 and OE-2 have larger stomata.

[0110] To further explore the reasons for the differences in drought tolerance among rice varieties, we analyzed the antioxidant activity and ABA content in rice leaves of Oscpn10a knockout mutants and OE lines. The results showed that compared with before drought treatment, the activities of SOD, POD, CAT, and MDA content in wild-type, Oscpn10a knockout, and OsCPN10a overexpression seedlings all increased after drought treatment, indicating that the rice's antioxidant system was activated. Compared with the wild-type under the same treatment, the MDA content of Oscpn10a-2 and Oscpn10a-4 seedlings was significantly increased (P < 0.05), by 24.19% and 36.23%, respectively, while the MDA content of OE-1 and OE-2 was significantly decreased, by 18.66% and 21.66%, respectively; the activities of SOD, POD, and CAT in Oscpn10a-2 and Oscpn10a-4 seedlings were significantly decreased. (P < 0.05). Specifically, the activities of SOD, POD, and CAT in Oscpn10a-2 seedlings decreased by 15.61%, 25.12%, and 15.77%, respectively; those in Oscpn10a-4 seedlings decreased by 15.09%, 23.77%, and 25.98%, respectively; while the activities of SOD, POD, and CAT in the leaves of OsCPN10a overexpressing plants were similar to those in the wild type. Furthermore, ABA content was measured after 3 and 5 days of drought stress. The ABA content in wild-type and OsCPN10a plants recovered more quickly, while the ABA content in Oscpn10a recovered more slowly.

[0111] In summary, overexpression of OsCPN10a can mitigate drought by reducing stomatal water loss. Considering the active participation of OsCPN10a in the ABA response, we hypothesize that OsCPN10a may play a positive regulatory role in the ABA-dependent drought stress response.

[0112] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of OsCPN10a gene in regulating rice seed vigor, grain type and drought tolerance, characterized in that, The amino acid sequence of the protein encoded by the OsCPN10a gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the OsCPN10a gene is shown as SEQ ID NO. 2; the regulation of seed kernel type is the regulation of seed kernel length, kernel width and 1000-grain weight.

2. Use according to claim 1, characterized in that, The regulation of seed activity is the regulation of seed germination rate, germination potential and germination index.

3. Use according to claim 1, characterized in that, The gene knockout is to take OsCPN10a as a target gene, adopt a CRISPR / Cas9 gene editing system, construct a knockout vector of the target gene, transform into rice, and cultivate the rice.

4. Use according to claim 3, characterized in that, The construction method of the knockout vector is: Select a target and design gRNA, perform PCR amplification with pCBC-MT1T2 plasmid as a template, recover it, and then use T4 ligase to connect it with pHUE411 vector to obtain a connection product, transform it into E. coli, extract the plasmid, and then perform sequencing verification.

5. Use according to claim 4, characterized in that, The gRNA sequence is shown as SEQ ID NO. 3 or 4.

6. Use according to claim 1, characterized in that, The gene overexpression is to take OsCPN10a as a target gene, adopt a seamless cloning and homologous recombination method to connect the target gene with a vector to construct an overexpression vector, transform into rice, and cultivate the rice.

7. Use according to claim 6, characterized in that, The construction method of the gene overexpression vector is: Design upper and lower stream primers for the OsCPN10a gene to perform PCR amplification, take HindIII and KpnI as enzyme cutting sites to cut the binary vector pRHVcGFP and the amplification product, and then connect the target fragment with the linearized vector through homologous recombination to obtain a recombination vector.

8. Use according to claim 7, characterized in that, The sequence of the upper stream primer is shown as SEQ ID NO. 11, and the sequence of the lower stream primer is shown as SEQ ID NO.

12.

9. Use of the OsCPN10a gene in breeding drought-resistant rice varieties, characterized in that, The nucleotide sequence of the OsCPN10a gene is shown as SEQ ID NO.

2.

10. A method of breeding a drought tolerant rice variety, characterized in that, Transform the expression vector containing the OsCPN10a gene into the target plant to perform overexpression, and obtain the drought-resistant rice; the nucleotide sequence of the OsCPN10a gene is shown as SEQ ID NO.

2. The nucleotide sequence of the OsCPN10a gene is shown as SEQ ID NO. 2.

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